Re: [ccp4bb] Help with Optimizing Crystals
Hi. Thanks for all of the helpful advice. Below is a summary of the suggestions, along with some things that I have tried and the results thus far. 1) Make sure that the crystals are protein and not salt. My crystals absorb Izit dye well and shooting some initial crystals did not produce any diffraction. I have yet to see them on a gel though, possibly due to too few crystals run, loss of crystals while washing, etc. 2) Try streak seeding using the hit conditions as well as new conditions. Streak seeding using two hit conditions and half of the protein concentration have thus far only produced crystals that are smaller than the initial crystals used to make the seed stock. This indicates that self nucleation is still occurs. I will try significantly lowering the precipitant conc. as well. Seeding into a few random conditions has produced some crystals in new conditions, but they look the same or worse that the previous ones. 3) Make sure that the protein is very pure. Include an additional purification step to your purification scheme, try thermal denaturation of contaminating proteins, crystallize out protein and the redissolve the crystals and use to set up new trays. I will try some of these. 4) Modify the protein to make it more amendable to crystallization. Try truncations, mutations, methylate lysines. I tried the FL version from two different organisms and neither crystallizes. When enzymatically proteolyzed, the protein from one organism crystallizes. However, when proteins containing similar truncations are purified recombinately, a ladder of bands appears after the major one, indicating protein instability. I have not tried any mutations or methylation yet. Thanks for all of the helpful suggestions, and if there are any more, please let me know. Matt On Wed, Oct 27, 2010 at 9:28 AM, Annie Hassell annie.m.hass...@gsk.comwrote: Matt— You might want to try heating your protein to get rid of unfolded/improperly folded protein. We have used 37C for 10 min with good success, but a time course at different temperatures is the best way to determine which parameters are optimum for your protein. Heat—chill it on ice—centrifuge-- then set up your crystallization trays. It’s a pretty quick test to see if this will work for your protein. Do you have any ligands for your protein? These have often been the key to getting good crystals in our lab. If you do have good ligands, you may want to express and/or purify your protein in the presence of these compounds. Good Luck! annie *From:* CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] *On Behalf Of *Jürgen Bosch *Sent:* Tuesday, October 26, 2010 5:46 PM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* Re: [ccp4bb] Help with Optimizing Crystals Hi. Here is some additional information. 1. The purification method that I used included Ni, tag cleavage, and SEC as a final step. I have tried samples from three different purification batches that range in purity, and even the batch with the worst purity seems to produce crystals. Resource Q ? two or more species perhaps ? Does it run as a monomer dimer multimer on your SEC ? 2. The protein is a proteolyzed fragment since the full length version did not crystallize. Mutagenesis and methylation, however, may be techniques to consider since the protein contains quite a few lysines. 3. There are not any detergents in the buffer, so these are not detergent crystals. The protein buffer just contains Tris at pH 8, NaCl, and DTT. 4. Some experiments that I have done thus far seem to suggest that the crystals are protein. Izit dye soaks well into the crystals, and the few crystals that I shot previously did not produce any diffraction pattern whatsoever. However, I have had difficulty seeming them on a gel and they are a bit tough to break. Do they float or do they sink quickly when you try to mount them ? 5. I tried seeding previously as follows: I broke some crystals, made a seed stock, dipped in a hair, and did serial streak seeding. After seeding, I usually saw small disks or clusters along the path of the hair but nothing larger or better looking. I also had one more question. Has anyone had an instance where changing the precipitation condition or including an additive improved diffraction but did not drastically change the shape of the protein? If so, I may just try further optimization with the current conditions and shoot some more crystals. The additive screen from Hampton is not bad and can make a big difference. A different topic is it a direct cryo what you are using as a condition ? If not what do you use a s a cryo ? Have you tried the old-fashioned way of shooting at crystals at room temperature using capillaries (WTHIT ?) You might be killing your crystal by trying to cryo it is what I'm trying to say here. Jürgen Thanks for all the helpful advice thus far, Matt
Re: [ccp4bb] Help with Optimizing Crystals
Matt- You might want to try heating your protein to get rid of unfolded/improperly folded protein. We have used 37C for 10 min with good success, but a time course at different temperatures is the best way to determine which parameters are optimum for your protein. Heat-chill it on ice-centrifuge-- then set up your crystallization trays. It's a pretty quick test to see if this will work for your protein. Do you have any ligands for your protein? These have often been the key to getting good crystals in our lab. If you do have good ligands, you may want to express and/or purify your protein in the presence of these compounds. Good Luck! annie From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Jürgen Bosch Sent: Tuesday, October 26, 2010 5:46 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Help with Optimizing Crystals Hi. Here is some additional information. 1. The purification method that I used included Ni, tag cleavage, and SEC as a final step. I have tried samples from three different purification batches that range in purity, and even the batch with the worst purity seems to produce crystals. Resource Q ? two or more species perhaps ? Does it run as a monomer dimer multimer on your SEC ? 2. The protein is a proteolyzed fragment since the full length version did not crystallize. Mutagenesis and methylation, however, may be techniques to consider since the protein contains quite a few lysines. 3. There are not any detergents in the buffer, so these are not detergent crystals. The protein buffer just contains Tris at pH 8, NaCl, and DTT. 4. Some experiments that I have done thus far seem to suggest that the crystals are protein. Izit dye soaks well into the crystals, and the few crystals that I shot previously did not produce any diffraction pattern whatsoever. However, I have had difficulty seeming them on a gel and they are a bit tough to break. Do they float or do they sink quickly when you try to mount them ? 5. I tried seeding previously as follows: I broke some crystals, made a seed stock, dipped in a hair, and did serial streak seeding. After seeding, I usually saw small disks or clusters along the path of the hair but nothing larger or better looking. I also had one more question. Has anyone had an instance where changing the precipitation condition or including an additive improved diffraction but did not drastically change the shape of the protein? If so, I may just try further optimization with the current conditions and shoot some more crystals. The additive screen from Hampton is not bad and can make a big difference. A different topic is it a direct cryo what you are using as a condition ? If not what do you use a s a cryo ? Have you tried the old-fashioned way of shooting at crystals at room temperature using capillaries (WTHIT ?) You might be killing your crystal by trying to cryo it is what I'm trying to say here. Jürgen Thanks for all the helpful advice thus far, Matt
[ccp4bb] Help with Optimizing Crystals
Hello. I have obtained disk shaped crystals of a protein that I am working on. I got hits in about 10 different conditions, with a few common precipitants and pHs, and I have optimized two conditions so far. In the optimized conditions, the crystals appear overnight, usually surrounded by or hiding under heavy precipitant. Under the best conditions, I get what I would describe as single disks, some of which are of decent size and very round, that rotate light very well. Sub-optimal conditions can give small to large crystal clusters. I shot the large disk crystals grown from one conditions at the synchrotron. but they do not diffract. I was wondering if anyone had any advice about optimizing these crystals in order to get them to diffract better? As mentioned before, I have only tried optimizing a few of the hit conditions (varying precipitant conc., pH, etc.), but crystals from all of the hits look the same: always round disks or disk clusters. This leads me to believe that optimized conditions of the other hits will produce similar results as before. Would it be worthwhile to try optimizing these conditions as well? I have also tried seeding, which just produces a lot of clusters, and an additive screen. Some of the additives help to produce larger crystals, but again I always get single or disk clusters. Any advice would be helpful. Thanks, Matt
Re: [ccp4bb] Help with Optimizing Crystals
Hi Matt, You'll probably get many different answers to a question like this, but what I would do is go back to your protein and make different constructs; chop off termini, surface mutations etc, maybe cleave off the tag. Of course more screening and optimization might work, but my sense is that since you get many hits pretty easily that however don't diffract, there may be something on the protein level that needs correcting. Good luck, Bert On 10/26/10 4:23 PM, Matthew Bratkowski mab...@cornell.edu wrote: Hello. I have obtained disk shaped crystals of a protein that I am working on. I got hits in about 10 different conditions, with a few common precipitants and pHs, and I have optimized two conditions so far. In the optimized conditions, the crystals appear overnight, usually surrounded by or hiding under heavy precipitant. Under the best conditions, I get what I would describe as single disks, some of which are of decent size and very round, that rotate light very well. Sub-optimal conditions can give small to large crystal clusters. I shot the large disk crystals grown from one conditions at the synchrotron. but they do not diffract. I was wondering if anyone had any advice about optimizing these crystals in order to get them to diffract better? As mentioned before, I have only tried optimizing a few of the hit conditions (varying precipitant conc., pH, etc.), but crystals from all of the hits look the same: always round disks or disk clusters. This leads me to believe that optimized conditions of the other hits will produce similar results as before. Would it be worthwhile to try optimizing these conditions as well? I have also tried seeding, which just produces a lot of clusters, and an additive screen. Some of the additives help to produce larger crystals, but again I always get single or disk clusters. Any advice would be helpful. Thanks, Matt
Re: [ccp4bb] Help with Optimizing Crystals
First piece of advice I have is to shove them in the beam and see what happens. A few days ago we got high-resolution data from crystals that are shaped like eggs. No edges on them whatsoever. In the past, saucer-shaped crystals diffracted to 2A whereas their hexagonal 'perfect' cousins (grown from a different PEG, if memory serves) had Cheeseburger-strength diffraction. Secondly, if ordinary optimization attempts repeatedly fail, it may be time for protein optimization, e.g. proteolysis, mutagenesis, methylation and so forth :) Artem On Tue, Oct 26, 2010 at 3:23 PM, Matthew Bratkowski mab...@cornell.eduwrote: Hello. I have obtained disk shaped crystals of a protein that I am working on. I got hits in about 10 different conditions, with a few common precipitants and pHs, and I have optimized two conditions so far. In the optimized conditions, the crystals appear overnight, usually surrounded by or hiding under heavy precipitant. Under the best conditions, I get what I would describe as single disks, some of which are of decent size and very round, that rotate light very well. Sub-optimal conditions can give small to large crystal clusters. I shot the large disk crystals grown from one conditions at the synchrotron. but they do not diffract. I was wondering if anyone had any advice about optimizing these crystals in order to get them to diffract better? As mentioned before, I have only tried optimizing a few of the hit conditions (varying precipitant conc., pH, etc.), but crystals from all of the hits look the same: always round disks or disk clusters. This leads me to believe that optimized conditions of the other hits will produce similar results as before. Would it be worthwhile to try optimizing these conditions as well? I have also tried seeding, which just produces a lot of clusters, and an additive screen. Some of the additives help to produce larger crystals, but again I always get single or disk clusters. Any advice would be helpful. Thanks, Matt
Re: [ccp4bb] Help with Optimizing Crystals
You did check on a gel that they are indeed your protein ? If you have sufficient amounts available try digesting it with various proteases and see if you can identify a stable fragment. A less radical approach, which might not be accessible to you, you could screen your protein for alternative buffer conditions using DSF and then pick a condition under which it seems to be very stable according to its melting temperature in the buffer. You've spared us the details of your purification procedure, maybe a polishing step at the end with a SEC might do wonders. Jürgen - Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ On Oct 26, 2010, at 4:23 PM, Matthew Bratkowski wrote: Hello. I have obtained disk shaped crystals of a protein that I am working on. I got hits in about 10 different conditions, with a few common precipitants and pHs, and I have optimized two conditions so far. In the optimized conditions, the crystals appear overnight, usually surrounded by or hiding under heavy precipitant. Under the best conditions, I get what I would describe as single disks, some of which are of decent size and very round, that rotate light very well. Sub-optimal conditions can give small to large crystal clusters. I shot the large disk crystals grown from one conditions at the synchrotron. but they do not diffract. I was wondering if anyone had any advice about optimizing these crystals in order to get them to diffract better? As mentioned before, I have only tried optimizing a few of the hit conditions (varying precipitant conc., pH, etc.), but crystals from all of the hits look the same: always round disks or disk clusters. This leads me to believe that optimized conditions of the other hits will produce similar results as before. Would it be worthwhile to try optimizing these conditions as well? I have also tried seeding, which just produces a lot of clusters, and an additive screen. Some of the additives help to produce larger crystals, but again I always get single or disk clusters. Any advice would be helpful. Thanks, Matt
Re: [ccp4bb] Help with Optimizing Crystals
Seeding! Make seeds, rescreen with seeds. Look in many former ccp4bb posts for references about this. Jacob - Original Message - From: Jürgen Bosch To: CCP4BB@JISCMAIL.AC.UK Sent: Tuesday, October 26, 2010 3:47 PM Subject: Re: [ccp4bb] Help with Optimizing Crystals You did check on a gel that they are indeed your protein ? If you have sufficient amounts available try digesting it with various proteases and see if you can identify a stable fragment. A less radical approach, which might not be accessible to you, you could screen your protein for alternative buffer conditions using DSF and then pick a condition under which it seems to be very stable according to its melting temperature in the buffer. You've spared us the details of your purification procedure, maybe a polishing step at the end with a SEC might do wonders. Jürgen - Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ On Oct 26, 2010, at 4:23 PM, Matthew Bratkowski wrote: Hello. I have obtained disk shaped crystals of a protein that I am working on. I got hits in about 10 different conditions, with a few common precipitants and pHs, and I have optimized two conditions so far. In the optimized conditions, the crystals appear overnight, usually surrounded by or hiding under heavy precipitant. Under the best conditions, I get what I would describe as single disks, some of which are of decent size and very round, that rotate light very well. Sub-optimal conditions can give small to large crystal clusters. I shot the large disk crystals grown from one conditions at the synchrotron. but they do not diffract. I was wondering if anyone had any advice about optimizing these crystals in order to get them to diffract better? As mentioned before, I have only tried optimizing a few of the hit conditions (varying precipitant conc., pH, etc.), but crystals from all of the hits look the same: always round disks or disk clusters. This leads me to believe that optimized conditions of the other hits will produce similar results as before. Would it be worthwhile to try optimizing these conditions as well? I have also tried seeding, which just produces a lot of clusters, and an additive screen. Some of the additives help to produce larger crystals, but again I always get single or disk clusters. Any advice would be helpful. Thanks, Matt *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Help with Optimizing Crystals
Hi. Here is some additional information. 1. The purification method that I used included Ni, tag cleavage, and SEC as a final step. I have tried samples from three different purification batches that range in purity, and even the batch with the worst purity seems to produce crystals. 2. The protein is a proteolyzed fragment since the full length version did not crystallize. Mutagenesis and methylation, however, may be techniques to consider since the protein contains quite a few lysines. 3. There are not any detergents in the buffer, so these are not detergent crystals. The protein buffer just contains Tris at pH 8, NaCl, and DTT. 4. Some experiments that I have done thus far seem to suggest that the crystals are protein. Izit dye soaks well into the crystals, and the few crystals that I shot previously did not produce any diffraction pattern whatsoever. However, I have had difficulty seeming them on a gel and they are a bit tough to break. 5. I tried seeding previously as follows: I broke some crystals, made a seed stock, dipped in a hair, and did serial streak seeding. After seeding, I usually saw small disks or clusters along the path of the hair but nothing larger or better looking. I also had one more question. Has anyone had an instance where changing the precipitation condition or including an additive improved diffraction but did not drastically change the shape of the protein? If so, I may just try further optimization with the current conditions and shoot some more crystals. Thanks for all the helpful advice thus far, Matt
Re: [ccp4bb] Help with Optimizing Crystals
Hi. Here is some additional information. 1. The purification method that I used included Ni, tag cleavage, and SEC as a final step. I have tried samples from three different purification batches that range in purity, and even the batch with the worst purity seems to produce crystals. Resource Q ? two or more species perhaps ? Does it run as a monomer dimer multimer on your SEC ? 2. The protein is a proteolyzed fragment since the full length version did not crystallize. Mutagenesis and methylation, however, may be techniques to consider since the protein contains quite a few lysines. 3. There are not any detergents in the buffer, so these are not detergent crystals. The protein buffer just contains Tris at pH 8, NaCl, and DTT. 4. Some experiments that I have done thus far seem to suggest that the crystals are protein. Izit dye soaks well into the crystals, and the few crystals that I shot previously did not produce any diffraction pattern whatsoever. However, I have had difficulty seeming them on a gel and they are a bit tough to break. Do they float or do they sink quickly when you try to mount them ? 5. I tried seeding previously as follows: I broke some crystals, made a seed stock, dipped in a hair, and did serial streak seeding. After seeding, I usually saw small disks or clusters along the path of the hair but nothing larger or better looking. I also had one more question. Has anyone had an instance where changing the precipitation condition or including an additive improved diffraction but did not drastically change the shape of the protein? If so, I may just try further optimization with the current conditions and shoot some more crystals. The additive screen from Hampton is not bad and can make a big difference. A different topic is it a direct cryo what you are using as a condition ? If not what do you use a s a cryo ? Have you tried the old-fashioned way of shooting at crystals at room temperature using capillaries (WTHIT ?) You might be killing your crystal by trying to cryo it is what I'm trying to say here. Jürgen Thanks for all the helpful advice thus far, Matt
Re: [ccp4bb] Help with Optimizing Crystals
Janet Newman wrote a review on improving diffraction a few years back: http://dx.doi.org/10.1107/S0907444905032130 it is open access. Probably the most underappreciated aspect of diffraction is the purity of the protien. This is because impurities like slightly misfolded versions of your native structure can genreally still find their way into the lattice. These are defects, and the distorition they create will push thousands of other unit cells out of place. So, 95%, or even 99% purity can be not enough. Getting the molecule to the point where it is the brightest band on the gel is generally enough to screen for crystallization conditions, but it is not uncommon for crystals from un-clean protein to diffract poorly. My advice: try adding a column to your purification protocol. Or, better yet, try fractional recrystallization. This is where you use your crystallization condition to crash out your entire stock, spin down the crystals, redissolve them, and then maybe do this a few times in a row. Yes, you loose a lot of material, but the stuff you lost is stuff that doesn't crystallize anyway. -James Holton MAD Scientist On Tue, Oct 26, 2010 at 2:31 PM, Matthew Bratkowski mab...@cornell.eduwrote: Hi. Here is some additional information. 1. The purification method that I used included Ni, tag cleavage, and SEC as a final step. I have tried samples from three different purification batches that range in purity, and even the batch with the worst purity seems to produce crystals. 2. The protein is a proteolyzed fragment since the full length version did not crystallize. Mutagenesis and methylation, however, may be techniques to consider since the protein contains quite a few lysines. 3. There are not any detergents in the buffer, so these are not detergent crystals. The protein buffer just contains Tris at pH 8, NaCl, and DTT. 4. Some experiments that I have done thus far seem to suggest that the crystals are protein. Izit dye soaks well into the crystals, and the few crystals that I shot previously did not produce any diffraction pattern whatsoever. However, I have had difficulty seeming them on a gel and they are a bit tough to break. 5. I tried seeding previously as follows: I broke some crystals, made a seed stock, dipped in a hair, and did serial streak seeding. After seeding, I usually saw small disks or clusters along the path of the hair but nothing larger or better looking. I also had one more question. Has anyone had an instance where changing the precipitation condition or including an additive improved diffraction but did not drastically change the shape of the protein? If so, I may just try further optimization with the current conditions and shoot some more crystals. Thanks for all the helpful advice thus far, Matt
